PCR for more than 5 kb - (Sep/04/2005 )

dear all..

i want to know how can we PCR DNA bands more than 5 Kb in size?

any idea?

thanks

You have to add 1 min elongation time per kb to give the polymerase time to finish.

You should also be aware that using a polymerase without proofreading activity will result in several mutations per product (that might be relevant if you want to clone it). On the other hand proofreading enzymes don't handle long elongations quite so well as Taq.

The best option might be to go for a polymerase mix. I'm sure I've seen special polymerase mixes for longer products somewhere.

LeserattePD

Hi,

Roche sells a "Expand long template system". It contains a special enzyme mix. Worked well for me. Alternatively, you could mix Taq and Pfu by yourself.

Susan

It is better to use Polymerase mix.

Many commerical companies sell this kind of enzyme mix and show the maximun length of the PCR products.
Most of them can be up to 10-15kb.

first of all,thanks for ur attentions and ideas..

i read about LA taq.and there are difference between LA takara taq and Takara taq.which is better?

say if i got repeated sequence in the templates,can i still proceed the amplification using the taq?

thanks..